Photoreceptor degenerations lead to visual impairment and blindness. Since human retinas are incapable of regeneration, cell replacement therapy is an attractive approach to restore lost photoreceptors. Of particular interest is programming human embryonic stem (hES) cells into cone photoreceptors that can be used to restore high acuity daytime (photopic) vision in diseased retinas after transplant. To implement this strategy requires knowledge of the factors that control cone development, the conditions permissive for cone transplantation into adult retinas, and whether hES cell derived cones can restore photopic vision in diseased retinas. We will address these problems in the following specific aims: 1. To determine how cell extrinsic factors control cone photoreceptor specification and differentiation. Cell non-autonomous effects can control the numbers of each cell type in the retina;but how do they regulate cones? To test this, we will treat dissociated retinal progenitors with 6 candidate factors. To specifically label cones, retinal progenitors from M-opsin:GFP and thyroid hormone 02 knock-in (TR[52) mouse retinas will be collected. Dissociated cells will be labeled with BrdU, and treated with candidate factors for one week. The effects of these extrinsic factors on cone fate and differentiation will be quantified. 2. To determine the donor cell conditions that promote efficient transplantation of functional cones in adult retinas. Previous data suggests postmitotic, undifferentiated cells integrate best into adult retinas. To test this for cones, we will purify cones from TRB2 knock-in mice by flow cytometry and subretinally transplant them into wild-type mice. After 2 weeks, retinas will be scored for transplanted cones. The extent of integration at each time-point will be compared to determine what stages are permissive for cone transplantation. 3. To restore photopic vision in diseased retinas by transplantation of cones derived from hES cells. Our laboratory has developed a protocol to convert hES cells into retinal progenitor cells. To test whether these cells can restore photopic vision we will treat hES cells with extrinsic factors to enrich for cones. Next, we will transplant cone enriched hES cells into adult Crx null mice, which have no functional photoreceptors. We will measure photopic vision restoration 2-6 weeks later by electrophysiology and histology.